Maeder, E.

Abstract BACKGROUND Intraductal papillary mucinous neoplasm (IPMN) is one of the two most common precursor lesions leading to the development of pancreatic ductal adenocarcinoma (PDA). IPMNs comprise a heterogeneous group of tumors with a wide range of grades and histotypes, and the emergence of single-cell RNA sequencing (RNA-seq) and multiplex digital spatial profiling have characterized unique cell populations, including dysplastic epithelial and immune cells, within the heterogeneous tumor microenvironment that carry signature gene expressions, which could be used as markers for disease progression. However, it is not clear on the heterogeneity of the epithelial and immune response of the evolution during progression of IPMN to PDA. OBJECTIVE Combining nuclear GSK-3β with oncogenic KRas mice (referred to as KNGC – KRas, nuclear GSK-3β, Cre) resulted in the development of IPMN. We have found that 4-week-old KNGC mice show progressive desmoplasia and ductal lesion development with increased M2-like macrophage infiltration. Interestingly, CX3CL1 (fractalkine), a chemokine involved in the recruitment of CX3CR1-expressing monocytes and M2-like macrophage conversion in various disease models is expressed by IPMN progenitor ductal cells. We aim to investigate expression and contribution of CX3CL1-CX3CL1 axis in the development and progression of IPMN. METHODS We utilized a newly generated CX3CL1-mCherry and well-established CX3CR1-GFP knockin/knockout mouse for crossbreeding with KNGC and KGC (KRas, GNas, Cre) for lineage tracing and function analysis by immunofluorescent staining, flow cytometry and CyTOF. RESULTS Both KNGC- and KGC-3LmCherry/+ mice harbor a large population of CX3CL1-mCherry/EPCAM+ epithelial cells as compared to 3LmCherry/+ and wildtype (WT) control mice. In KNGC and KGC mice, we show that there was a unique population of macrophages (CD45+ CD11b+ Ly6G- F4/80+ Ly6c- ) with significantly higher expression of CX3CR1 (CX3CR1Hi) that was not seen in WT mice. Significantly, Knockout of CX3CR1 in KNGC-3RGFP/GFP animals had a paucity of M2-like macrophages, reduced T regulatory cells (Tregs), substantially reduced desmoplasia and impaired development of IPMN. CONCLUSIONS The study provides important knowledge regarding the role of CX3CL1-CX3CR1 axis in recruitment and generation of M2-like macrophages, and their impact on the initiation of the immune/stroma microenvironment facilitating IPMN development. Further investigations using these models have the potential to identify new biomarkers for risk stratification and therapeutic intervention. Citation Format: Li Ding, Kaely Roeck, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Esther Rodman, Mark Maynes, Bibo Zhu, Aaron Johnson, Jie Sun, Li Hu, Daniel Billadeau. Uncoupling CX3CL1-CX3CR1 Axis in IPMN Development Through Transgenic Mouse Models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A064.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a dismal 5-year survival rate, highlighting the urgent need to improve drug response and overcome primary or acquired (secondary) resistance to conventional chemotherapy. Glycogen synthase kinase 3β (GSK-3β) is a target in PDAC and the GSK-3 inhibitor, elraglusib, has been shown to improve chemosensitivity of PDAC and survival in preclinical cancer models. In addition, data from phase II clinical trials in patients with previously untreated metastatic pancreatic cancer is demonstrating preliminary evidence of clinical benefit in patients receiving a combination of gemcitabine/nab-paclitaxel. While this early evidence is highly encouraging, the biological determinants of elraglusib response are unknown. Using genome wide CRISPR/Cas9 library screening, we identify aurora kinase A (AURKA), an oncogenic kinase with major roles in mitosis, as a critical regulator for elraglusib sensitivity. Elraglusib treatment activates AURKA by inducing a G2/M cell cycle arrest. We show that knocking out AURKA reduces the phosphorylation of polo like kinase 1 (PLK1), arresting cells in mitosis, and leading to mitotic catastrophe and significant apoptosis after elraglusib treatment. Strikingly, the AURKA inhibitor, alisertib, works synergistically with elraglusib to inhibit PDAC cell line growth and induce apoptosis. In summary, our results demonstrate that targeting AURKA is an effective approach to sensitize PDAC tumors to elraglusib. Citation Format: Li Ding, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Daniel Schmitt, Andrew Mazar, Hu Li, Daniel Billadeau. Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4658.

ABSTRACT Natural killer (NK) cells have the ability to lyse other cells through the release of lytic granules (LGs). This is in part mediated by the small GTPase Rab27a, which was first identified to play a crucial role in degranulation through the study of individuals harboring mutations in the gene encoding Rab27a. However, the guanine nucleotide exchange factor (GEF) regulating the activation of Rab27a in cytotoxic lymphocytes was unknown. Here, we show that knockout of MADD significantly decreased the levels of GTP-bound Rab27a in both resting and stimulated NK cells, and MADD-deficient NK cells and CD8+ T cells displayed severely reduced degranulation and cytolytic ability, similar to that seen with Rab27a deficiency. Although MADD colocalized with Rab27a on LGs and was enriched at the cytolytic synapse, the loss of MADD did not impact Rab27a association with LGs nor their recruitment to the cytolytic synapse. Together, our results demonstrate an important role for MADD in cytotoxic lymphocyte killing.